Eva-Christina Müller

PROTEOMICS IN HUMAN DISEASE : CANCER, HEART AND INFECTIOUS DISEASES

In recent years, genomics has increased the understanding of many diseases. Proteomics is a rapidly growing research area that encompasses both genetic and environmental factors. The protein composition represents the functional status of a biological compartment. The five approaches presented here resulted in the detection of disease‐associated proteins. Calgranulin B was upregulated in colorectal cancer, and hepatoma‐derived aldose reductase‐like protein was reexpressed in a rat model during hepatocarcinogenesis. In these two investigations, attention was focused on one protein, obviously differing in amount, directly after two‐dimensional electrophoresis (2‐DE). Additional methods, such as enzyme activity measurements and immunohistochemistry, confirmed the disease association of the two candidates resulting from 2‐DE subtractive analysis. The following three investigations take advantage of the holistic potential of the 2‐DE approach. The comparison of 2‐DE patterns from dilated cardiomyopathy patients with those of controls revealed 25 statistically significant intensity differences, from which 12 were identified by amino acid analysis, Edman degradation or matrix‐assisted laser desorption/ionization‐mass spectrometry (MALDI‐MS). A human myocardial 2‐DE database was constructed, containing 3300 protein spots and 150 identified protein species. The number of identified proteins was limited by the capacity of our group, rather than by the principle of feasibility. Another field where proteomics proves to be a valuable tool in identifying proteins of importance for diagnosis is proteome analysis of pathogenic microorganisms such as Borrelia burgdorferi (Lyme disease) and Toxoplasma gondii (toxoplasmosis). Sera from patients with early or late symptoms of Lyme borreliosis contained antibodies of various classes against about 80 antigens each, containing the already described antigens OspA, B and C, flagellin, p83/100, and p39. Similarly, antibody reactivity to seven different marker antigens of T. gondii allowed differentiation between acute and latent toxoplasmosis, an important diagnostic tool in both pregnancy and immunosuppressed patients.

Biochemical and Proteomic Analysis of the Magnetosome Membrane in Magnetospirillum gryphiswaldense

ABSTRACT We analyzed the biochemical composition of the magnetosome membrane (MM) in Magnetospirillum gryphiswaldense. Isolated magnetosomes were associated with phospholipids and fatty acids which were similar to phospholipids and fatty acids from other subcellular compartments (i.e., outer and cytoplasmic membranes) but were present in different proportions. The binding characteristics of MM-associated proteins were studied by selective solubilization and limited proteolysis. The MM-associated proteins were further analyzed by various proteomic approaches, including one- and two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by Edman and mass spectrometric (electrospray ionization-mass spectrometry-mass spectrometry) sequencing, as well as capillary liquid chromatography-mass spectrometry-mass spectrometry of total tryptic digests of the MM. At least 18 proteins were found to constitute the magnetosome subproteome, and most of these proteins are novel for M. gryphiswaldense. Except for MM22 and Mms16, all bona fide MM proteins (MMPs) were encoded by open reading frames in the mamAB, mamDC, and mms6 clusters in the previously identified putative magnetosome island. Eight of the MMPs display homology to known families, and some of them occur in the MM in multiple homologues. Ten of the MMPs have no known homologues in nonmagnetic organisms and thus represent novel, magnetotactic bacterium-specific protein families. Several MMPs display repetitive or highly acidic sequence patterns, which are known from other biomineralizing systems and thus may have relevance for magnetite formation.

Exportin‐5‐mediated nuclear export of eukaryotic elongation factor 1A and tRNA

Transport of proteins and RNA into and out of the cell nucleus is mediated largely by a family of RanGTP‐binding transport receptors. Export receptors (exportins) need to bind RanGTP for efficient loading of their export cargo. We have identified eukaryotic elongation factor 1A (eEF1A) and tRNA as RanGTP‐dependent binding partners of exportin‐5 (Exp5). Exp5 stimulates nuclear export of eEF1A when microinjected into the nucleus of Xenopus laevis oocytes. Surprisingly, the interaction between eEF1A and Exp5 is dependent on tRNA that can interact directly with Exp5 and, if aminoacylated, recruits eEF1A into the export complex. These data suggested to us that Exp5 might support tRNA export. Indeed, not only the canonical tRNA export receptor, exportin‐t, but also Exp5 can drive nuclear export of tRNA. Taken together, we show that there exists an alternative tRNA export pathway which can be exploited to keep eEF1A out of the cell nucleus.

Phosphorylation by protein kinase CK2: a signaling switch for the caspase-inhibiting protein ARC.

Caspases play a central role in apoptosis, but their activity is under the control of caspase-inhibiting proteins. A characteristic of caspase-inhibiting proteins is direct caspase binding. It is yet unknown how the localization of caspase-inhibiting proteins is regulated and whether there are upstream signals controlling their function. Here we report that the function of ARC is regulated by protein kinase CK2. ARC at threonine 149 is phosphorylated by CK2. This phosphorylation targets ARC to mitochondria. ARC is able to bind to caspase-8 only when it is localized to mitochondria but not to the cytoplasm. Our results reveal a molecular mechanism by which a caspase-inhibiting protein requires phosphorylation in order to prevent apoptosis.

Protein-rRNA binding features and their structural and functional implications in ribosomes as determined by cross- linking studies

We have investigated protein‐rRNA cross‐links formed in 30S and 50S ribosomal subunits of Escherichia coli and Bacillus stearothermophilus at the molecular level using UV and 2‐iminothiolane as cross‐linking agents. We identified amino acids cross‐linked to rRNA for 13 ribosomal proteins from these organisms, namely derived from S3, S4, S7, S14, S17, L2, L4, L6, L14, L27, L28, L29 and L36. Several other peptide stretches cross‐linked to rRNA have been sequenced in which no direct cross‐linked amino acid could be detected. The cross‐linked amino acids are positioned within loop domains carrying RNA binding features such as conserved basic and aromatic residues. One of the cross‐linked peptides in ribosomal protein S3 shows a common primary sequence motif–the KH motif–directly involved in interaction with rRNA, and the cross‐linked amino acid in ribosomal protein L36 lies within the zinc finger‐like motif of this protein. The cross‐linked amino acids in ribosomal proteins S17 and L6 prove the proposed RNA interacting site derived from three‐dimensional models. A comparison of our structural data with mutations in ribosomal proteins that lead to antibiotic resistance, and with those from protein‐antibiotic cross‐linking experiments, reveals functional implications for ribosomal proteins that interact with rRNA.

Multiple pathways contribute to nuclear import of core histones

Nuclear import of the four core histones H2A, H2B, H3 and H4 is one of the main nuclear import activities during S‐phase of the cell cycle. However, the molecular machinery facilitating nuclear import of core histones has not been elucidated. Here, we investigated the pathways by which histone import can occur. First, we show that core histone import can be competed by the BIB (β‐like import receptor binding) domain of ribosomal protein L23a suggesting that histone import is an importin mediated process. Secondly, affinity chromatography on immobilized core histones revealed that several members of the importin β family of transport receptors are able to interact with core histones. Finally, we demonstrate that at least four known and one novel importin, importin 9, can mediate nuclear import of core histones into the nuclei of permeabilized cells. Our results suggest that multiple pathways of import exist to provide efficient nuclear uptake of these abundant, essential proteins.

Proteomics reveals open reading frames in Mycobacterium tuberculosis H37Rv not predicted by genomics

ABSTRACT Genomics revealed the sequence of 3924 genes of the H37Rv strain ofMycobacterium tuberculosis. Proteomics complements genomics in showing which genes are really expressed, and here we show the expression of six genes not predicted by genomics, as proved by two-dimensional electrophoresis and matrix-assisted laser desorption ionization and nano-electrospray mass spectrometry.

Identification of proteins from Mycobacterium tuberculosis missing in attenuated Mycobacterium bovis BCG strains

A proteome approach, combining high‐resolution two‐dimensional electrophoresis (2‐DE) with mass spectrometry, was used to compare the cellular protein composition of two virulent strains of Mycobacterium tuberculosis with two attenuated strains of Mycobacterium bovis Bacillus Calmette‐Guérin (BCG), in order to identify unique proteins of these strains. Emphasis was given to the identification of M. tuberculosis specific proteins, because we consider these proteins to represent putative virulence factors and interesting candidates for vaccination and diagnosis of tuberculosis. The genome of M. tuberculosis strain H37Rv comprises nearly 4000 predicted open reading frames. In contrast, the separation of proteins from whole mycobacterial cells by 2‐DE resulted in silver‐stained patterns comprising about 1800 distinct protein spots. Amongst these, 96 spots were exclusively detected either in the virulent (56 spots) or in the attenuated (40 spots) mycobacterial strains. Fifty‐three of these spots were analyzed by mass spectrometry, of which 41 were identified, including 32 M. tuberculosis specific spots. Twelve M. tuberculosis specific spots were identified as proteins, encoded by genes previously reported to be deleted in M. bovis BCG. The remaining 20 spots unique for M. tuberculosis were identified as proteins encoded by genes that are not known to be missing in M. bovis BCG.

Characterization of OPA1 isoforms isolated from mouse tissues

OPA1, a nuclear encoded mitochondrial protein causing autosomal dominant optic atrophy, is a key player in mitochondrial fusion and cristae morphology regulation. In the present study, we have compared the OPA1 transcription and translation products of different mouse tissues. Unlike in humans, we found only two exons (4b and 5b) to be involved in alternative splicing. The relative abundance of the resulting four different splice variants is tissue‐dependent. Proteolytic cleavage by mitochondrial processing peptidase generates two long forms, isoforms 1 and 7, which lead to three short forms representing the end products after further proteolytic processing. In contrast, isoforms 5 and 8 are directly processed into their corresponding short forms. Short form 1 molecules form 184 kDa dimers, whereas all other isoforms contribute to 285 kDa complexes. Coiled‐coil domains of the OPA1 protein specifically homo‐associate and may be involved in the formation of these complexes. Furthermore, the region encoded by exon 5b inhibits the self‐association of coiled‐coil domain‐I. Finally, our data pinpoint isoform 1 as the, by far, most abundant isoform in the nervous tissue. We postulate that manipulation of isoform 1 protein levels in relation to the other isoforms induces changes in the mitochondrial network in the cell and therefore, mutations affecting the level of functional isoform 1 could lead to devastating effects on retinal ganglion cells.

Biochemical analysis of proteasomes from mouse microglia: Induction of immunoproteasomes by interferon‐γ and lipopolysaccharide

The 20S proteasome is a multicatalytic threonine protease and serves to process peptides that are subsequently presented as antigenic epitopes by MHC class I molecules. In the brain, microglial cells are the major antigen presenting cells and they respond sensitive to pathologic events. We used cultured mouse microglia and a microglial cell line, the BV‐2 line, as a model to study the correlation between microglial activation parameters and structural plasticity of the 20S/26S proteasome. Lipopolysaccharide (LPS)‐ or interferon‐γ (IFN‐γ)‐stimulated microglia or BV‐2 cells exhibit properties of activated microglia such as high levels of TNFα and IL‐6 release. In response to IFN‐γ or LPS, three constitutive β subunits (β1/Delta, β2/MC14, β5/MB1) were replaced by the immunoproteasome subunits iβ1/LMP2, iβ2/MECL‐1, and iβ5/LMP7, indicating that activated microglia adapts its proteasomal subunit composition to the requirements of an optimized MHC class I epitope processing. Induction of immunoproteasomes in BV‐2 cells was solely provoked by IFN‐γ, but not by LPS. Moreover, LPS (but not IFN‐γ) triggered the expression of a novel protein of ≈50 kD as part of the proteasome activator PA700, that is the substrate‐recognizing and unfolding unit of the 26S proteasome. These results indicate that both the 20S core protease as well as the proteasome activator PA700 are targets of modulatory subunit replacements or transient association of regulatory components in the course of microglial activation. GLIA 29:355–365, 2000.